Cancer is actively avoided through the expression of numerous tumor-suppressor genes that regulate the cell division cycle and mediate interactions among cells. Studies of benign and malignant tumors have shown that cancer develops through a multi-step process where randomly accumulated changes either enhance the expression of proto-oncogenes or reduce the expression or function of tumor-suppressor and DNA repair genes. Somatic mutations account for some of these changes in tumor-suppressor and DNA repair genes. However, it has recently become apparent that epigenetic changes, such as DNA hypermethylation and hypomethylation, also play a large role in the development of cancer through inactivation or enhancement of tumor suppressors or proto-oncogenes. Hypermethylation of CpG promoter islands occurs at an early stage of cancer development and is found in virtually all tumors, making it potentially very useful as a diagnostic marker, allowing cancer to be noninvasively detected in the early stages when treatment is most effective. For example, hypermethylation of the promoter region of genes such as DAP kinase, p16, and MGMT has reportedly been detected in the sputum of smokers up to 3 years prior to the diagnosis of squamous cell lung carcinoma (Belinsky et al. (2006). Cancer Res. 66:3338-44, Palmisano et al. 2000. Cancer Res. 60:5954-8). Similarly, hypermethylation of a small panel of genes may be a valuable early detection indicator for non-small cell lung cancer. Hypermethylation of a small panel of genes was detected in the early stages of breast cancer but was not detected in normal or benign breast tissue Krassenstein et al. (2004). Clin Cancer Res. 10:28-32.
Methylation of cytosines in CpG islands is an early event in most cancers that leads to reduced expression of many genes, including tumor suppressor genes. Surveys of CpG island methylation in tumor cell DNA suggest that this epigenetic change is common enough to rival the impact of mutation in tumor progression. Early detection of abnormal methylation can lead to regular screening and early diagnosis when treatment is most effective. Early epigenetic changes are often detectable in blood serum or other bodily fluids (urine, sputum, saliva), not just in the tumor tissue itself, which means that epigenetic diagnostic testing can be done noninvasively using these fluids. Diagnostic tests based on CpG island methylation may also be utilized for drug development, identification of patient populations that will respond to these drugs and post treatment monitoring.
Recent improvements in the sensitivity of methylation detection and elucidation of methylation signatures for specific cancers have made it feasible to assay for tumors by sampling DNA from bodily fluids. Tumor cells release DNA into blood from a relatively early stage of the disease. From 3% to over 90% of the DNA in the blood of cancer patients has been found to be of tumor origin (Kim et al. (2004) J. Clin. Oncol. 22:2363-70. The development of PCR-based methylation assays has made it possible to detect the presence of tumors noninvasively from blood, sputum, and urine. In one study methylation detection was more sensitive than urine cytology in detecting aberrant premalignant cells. Clinical sensitivities (the proportion of confirmed cases detected) are typically low in surveys of blood samples using a single methylation marker. However, clinical specificity (the proportion of normal controls that test negative) from blood samples approaches 100%. The clinical sensitivities should increase with methylation tests that assess more than a single CpG island.
The appearance of abnormally methylated DNA in bodily fluids by itself does not help to pinpoint which organ is affected by a tumor. Surprisingly little information is needed to establish the tissue origin of a tumor. Numerous methylation screening studies have established methylation signatures; collections of methylated CpG islands that are strongly associated with cancers from particular organs. In one survey, aberrant methylation of 3 to 4 candidate CpG islands was sufficient to identify from 70-90% of 15 cancer types (Esteller et al. (2001) Cancer Res. 61:3225-9). Methylation profiles developed for primary tumors could be applied to tumor cell lines to accurately identify the tissue origin of their parent tumors (Graziano et al. (2004) Clin. Cancer Res. 10:2784). This result suggests that methylation signatures of different tumor types are not greatly affected by the selective pressures associated with growth in culture. The availability of methylation profiles should greatly enhance the ability to detect cancer in inaccessible organs through a simple blood test.
Detection of methylation in clinical samples would enable early detection of cancer. The development of simple and sensitive multiplex detection assays will allow small clinical samples to be profiled for the status of multiple CpG islands. This kind of information will be valuable in diagnosis and treatment.
Methods for Detecting DNA Methylation
A number of methods have been used to detect methylated-CpG (mCpG) in target DNA. The three primary methods in current use are detailed below.
Bisulfite Methods. The most commonly used methylation detection methods are based on bisulfite modification of DNA, resulting in deamination of cytosine residues to uracil while leaving the methylated cytosines unchanged. Upon PCR amplification, the methylated cytosine is copied to cytosine and uracil is copied to thymine. As a result, the retention of cytosine at a specific position indicates methylation. The modified DNA is then analyzed, e.g. by sequence analysis, methylation-specific PCR (MSP) (Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-26), or hybridization (e.g. to a microarray or blot). In MSP, a pair of methylation-specific oligonucleotide primers is added to the bisulfite-treated DNA and PCR is performed in order to amplify the target DNA. Fluorescence-based quantitative real-time PCR can also be performed on bisulfite-modified DNA (Eads et al. (2000) Nucl. Acids Res. 28:E32; Zeschnigk et al. (2004) Nucl. Acids Res. 32:e125).
Calibrated, fluorescence-based variants of MSP exploit real-time PCR to provide quantification of the amount of methylated DNA in a sample. An important underlying assumption of these PCR based methods is that the few CpG sites that are recognized by the primers/probes reflect the overall status of the target CpG island. While this is usually true for heavily methylated or completely unmethylated islands, partially methylated targets are probably not readily scored in methylated- or unmethylated-specific reactions.
An advantage of bisulfite modification is that it differentially marks methylated versus unmethylated sites allowing sequencing methods to detect methylation patterns. Sequencing of cloned bisulfite-treated DNA is the most commonly used method for methylation detection. It provides information on the success of the bisulfite treatment in addition to sampling a greater number of CpG sites than the MSP based methods. Due to its complexity and expense, however, bisulfite sequencing is better suited for marker discovery than clinical diagnostics. Bisulfite treatment destroys a large percentage of the input DNA, resulting in limited sensitivity and a requirement for large amounts of DNA. Quality control assessments of bisulfite treated DNA are necessary before performing a detection assay to avoid misleading results. There is a potential of false-positive results for MSP-based assays due to incomplete cytosine deamination during bisulfite treatment. Amplification of bisulfite treated DNA is affected by PCR bias favoring unmethylated DNA. While this problem can usually be corrected by optimizing primer annealing conditions, it may complicate primer design and testing. Template biases can be eliminated with the use of digital bisulfite-PCR. Dilution of the DNA sample to an average of less than one copy per reaction eliminates competition among templates. Individual molecules can be sequenced without biases introduced by cloning.
Commercial kits, reagents and systems employing bisulfite treatment for analyzing mCpG are available. Epigenetics (Berlin) offers two variants of the MethyLight assay, adaptations of quantitative real-time PCR, called Quantitative MethyLight (QM) and Heavy Methyl (HM). QM utilizes Taqman® probes to generate a fluorescent signal. During the course of amplification, the fluor is cleaved from the Taqman® probe resulting in fluorescence that can be detected in real-time (Wojdacz & Dobrovic (2007) Nucl. Acids Res. 35:e41). HM is an adaptation of QM in which blocker oligonucleotides are added to the reaction. These blocker oligonucleotides prevent amplification of unmethylated DNA, resulting in increased assay sensitivity (Cottrell et al. (2004) Nucl. Acids Res. 32:e10). Pyrosequencing® is also utilized for methylation quantification from bisulfite-modified DNA, as exemplified by the Pyro Q-CpG™ system from Biotage (Uppsala, Sweden; Tost et al. (2003) Biotechniques 35:152-56).
Although bisulfite modification is a widely used, the extensive DNA degradation it causes can introduce sampling errors when few molecules are long enough to be amplified (Ehrich et al. (2007) Nucl. Acids Res. 35:e29). Furthermore, the assays are time-consuming, require a harsh base denaturation step, and have a high-probability of false-positive results due to incomplete cytosine deamination during bisulfite treatment.
Methylation-Sensitive Restriction Enzyme Digestion Methods. A second type of method for detecting mCpG in DNA relies on differential cleavage by restriction endonucleases. DNA is treated with either a MSRE (methylation-sensitive restriction endonuclease) or a MDRE (methylation dependent restriction endonuclease), amplified and then analyzed by microarray or gel electrophoresis. MSREs such as HpaII and AciI cut a DNA sequence only if it is unmethylated. MDREs are restriction endonuclease that require methylation of a DNA sequence for cleavage. By treating a sample of DNA with either of these enzymes and subsequent comparison to a control sample, the methylation state of the DNA sample can be determined. If digestion of a specific DNA sample occurs after treatment with a MDRE, then the DNA can be assumed to be methylated. Conversely, if the DNA is uncut when treated with a MSRE, then the sample can be assumed to be methylated. By comparing the amount of cut versus uncut DNA, the level of methylation can be estimated. A common read-out for this type of methylation analysis is the subsequent amplification and fluorescent labeling of the digested DNA. The fragments can then be hybridized to a library microarray and analyzed or simply resolved by electrophoresis.
Commercially available restriction endonuclease-based systems include Orion's MethylScope, which utilizes a microarray read-out (Lippman et al. (2004) Nature 430:471-76), and MethyScreen, which employs quantitative real-time PCR (Ordway et al. (2006) Carcinogenesis 27:2409-23).
An advantage of MSRE/MDRE digestion is that no pre-treatment of the DNA is necessary, although it is often performed in conjunction with bisulfite treatment of DNA in a procedure called COBRA (Xiong & Laird (1997) Nucl. Acids Res. 25:2532-34). Some disadvantages with this procedure are that it is lengthy and is dependent on the presence of MSRE/MDRE recognition sequences within a target DNA. Furthermore, this approach is relatively inefficient, which can reduce the reliability of the results. The only CpG sites that are assessed are those within a small number of restriction enzyme recognition sites and status of those sites may not reflect the status of the entire CpG island in which the site reside. Incomplete digestion leads to frequent false positives, especially when cleavage reactions are subjected to a subsequent amplification step. Restriction endonuclease cleavage assays have poor sensitivity compared to bisulfite methods, such as MSP, allowing detection of not less than 10% methylated DNA in a sample (Singer-Sam et al. Nucleic Acids Res, 1990. 18:687; Yegnasubramanian et al. Nucleic Acids Res. (2006) 34:e19).
Chromatin Immunoprecitipation Methods. A third method that is commonly employed for detecting mCpG is chromatin immunoprecipitation (ChIP). Typically, cells are fixed, and then methylated DNA is immunoprecipitated by the use of antibodies specific for methyl binding proteins. The resulting DNA is amplified, labeled and analyzed by hybridization in a microarray assay. The advantages of this method are that the assay can be performed from live cells with little or no DNA purification required. The assay also has increased sensitivity, as unwanted and contaminant DNA are removed prior to analysis. However, the ChIP procedure is very time-consuming, involves several steps and requires expensive reagents. Some assays may take as long as five days to complete.
Methods using Methyl Binding Proteins. An alternative and more sensitive approach to separating methylated from unmethylated DNAs involves the use of methyl-CpG binding domain (MBD) proteins or antibodies against 5-methyl-C. MBD proteins have high affinity for methylated CpG sites and very low affinity for unmethylated DNA (Fraga et al. Nucleic Acids Res. (2003) 31:1765-74). Samples are incubated with immobilized MBD protein in a variety of formats (magnetic beads, columns, the walls of PCR tubes). Methylated DNA capture is usually followed by amplification of the captured DNA. MBD-based DNA detection has the major advantage that all of the methylated sites can contribute to binding, thereby allowing an entire island to be sampled for methyl-CpGs. This characteristic makes the binding assay less vulnerable to false negatives that affect MSP and restriction endonuclease-based assays when unmethylated sites in a partially methylated island correspond to priming/probe sites (Yegnasubramanian et al. Nucleic Acids Res. (2006) 34:e19). This situation is likely to be common in clinical samples containing early stage tumor cells that contain partially methylated CpG islands. MBD based binding assays are very sensitive, allowing detection of as little as 160 pg of methylated DNA (equivalent to ˜25 cells) or 1 methylated molecule in 500 unmethylated molecules (Gebhard et al. Nucleic Acids Res. (2006) 34:e8256). This is close to the sensitivity of MSP (1 methylated molecule/1,000 unmethylated molecules). The COMPARE MBD assay can be as sensitive as real-time MSP (1 methylated molecule/10,000 unmethylated molecules) by including digestion with HpaII (an MSRE) before the binding step. Cleavage of unmethylated DNAs at a location between PCR priming sites gives high sensitivity with DNA mixtures that contain artificially methylated DNAs that are fully methylated (Yegnasubramanian et al. Nucleic Acids Res. (2006) 34:e19). However this strategy could suffer the disadvantage associated with the use of restriction endonucleases in that some partially methylated islands will be scored as unmethylated in clinical samples (Yegnasubramanian, et al. supra).
Given the importance of CpG methylation in cancer development and progression, a rapid, reliable, and sensitive test for methylated CpG DNA would provide an important and useful tool for cancer screening.